Effect And Mechanism Study Of Harmaline In Suppressing Growth Of Esophageal Squamous Cell Carcinoma

Background: Esophageal squamous cell carcinoma(ESCC)is the most common type of esophageal cancer worldwide.Although clinical advances in diagnostic and therapeutic techniques,the 5-year survival rate of esophageal cancer patients is still less than 25%.Additionally,RTK inhibitors have been investigated to treat ESCC patients,but these inhibitors have shown limited responses.Therefore,identifying more effective targets against ESCC is necessary.Mammalian target of rapamycin(mTOR)is a crucial factor in cell growth and metabolism,and frequently activated in many types of cancer.There are two subtypes: mTORC1 and mTORC2.They have different structures and functions.The group they share is mTOR kinase.There are two phosphorylation sites in mTOR kinase: Ser2481,which was known as autophosphorylation site of mTOR,and Ser 2448,which can be actived by AKT.mTORC1 is a potent driver of cellular proliferation and potentiates the severity of tumor progression.Active mTORC2 phosphorylates Akt at the hydrophobic motif(HM)site serine 473 and then promote protein synthesis.There are some regulation between mTORC1 and mTORC2.On the one hand,mTORC1 can active its downstream p70S6 K,and then inhibit Dictor,which is a part of mTORC2.mTORC1 can inhibit mTORC2 activity in this way.On the other hand,mTORC2 can active AKT,and then AKT can phosphorylate mTORC1 in Ser2448.Maybe this mechanism can explain the reason why treated with mTORC1 inhibitor will case the activation of AKT.Nowadays,mTOR inhibitors have been tested in many clinical trials of various cancers,however,resistance mechanisms induced by those inhibitors achieved only modest efficacy in cancer treatments.Thus,novel therapeutic strategies with mTORC1/2 inhibitor deserved to be further researched.Harmaline is a natural occurrence β-carboline alkaloid that isolated from the seeds of Peganum harmala(1).Previous studies have reported that harmaline exhibits numerous clinical effects,including antiinflammation,antianalgesia,antipruritic effects,and antitumor effect.Although harmaline exhibited antitumor effects in gastric and liver cancer cells,the molecular mechanism of harmaline and its anticancer activities has not been investigated in ESCC.In present study,we report that harmaline is a novel mTOR inhibitor that suppresses ESCC growth in vitro and in vivo.Methods: 1.Study the effect of harmaline on esophagus cancer cell growth.(1)MTT assay.To detect the effect of different doses of harmaline(0 μM,20 μM,40 μM,60 μM)on ESCC cell lines proliferation.(2)Foci-formation assay.To detect several doses of harmaline(0 μM,20 μM,40 μM,60 μM)on ESCC cell lines foci formation.(3)Soft agar assay.To detect different doses of harmaline(0 μM,20 μM,40 μM,60 μM)effection on ESCC cell lines anchorage-independent cell growth.2.Study the effect of harmaline on ESCC cell cycle.(1)Cell cycle analysis.(2)Western Blot.To detect harmaline effection in cell cycle related protein expression.3.Study the molecular target of harmaline in ESCC cell lines.(1)Kinase profiling service.To screen kinases,which was infected by harmaline.(2)Western Blot.To detect different kinases expression in JB6 cells after treated with harmaline.(3)In vitro kinase assay.To detect the activity of mTOR after treating with different concentration of harmaline.4.Study the expression and effect of mTOR in ESCC cell lines.(1)Western Blot.To compare the expression of mTOR in SHEE and ESCC cell lines and verify the effect of shmTOR.(2)MTT,Foci-formation and Soft agar.To study the growth of shmTOR ESCC cell lines.5.Explore whether the inhibit effect of harmaline in ESCC cell lines depends on the expression of mTOR.(1)MTT assay.To detect the effect of different doses of harmaline(0 μM,20 μM,40 μM,60 μM)on shmTOR cell lines proliferation.(2)Foci-formation assay.To detect several doses of harmaline(0 μM,20 μM,40 μM,60 μM)on shmTOR cell lines foci formation.(3)Soft agar assay.To detect effect of different doses of harmaline(0 μM,20 μM,40 μM,60 μM)on shmTOR cell lines anchorage-independent cell growth.6.Explore whether harmaline can inhibit the tumor growth in ESCC PDX model.(1)Build PDX model,give mice 100 mg/kg harmaline by oral from Monday to Friday,and give same volume of solvent for control group mice.Every week detect the tumor volume and bodyweight for each mice.(2)IHC to detect the expression of KI-67 in diferent group tumor.(3)Western Blot to compare the target molecular expression in treatment group and control group.(4)HE staining to detect the strucuture of liver,spleen,kidney in group and control group.(5)Detect ALT and AST level in the blood of group and control group to refelect liver founction in group and control group.Results: 1.(1),(2)and(3)results showed that harmaline can inhibit ESCC cell proliferation,foci-formation and clones in a dose dependent manner(P< 0.05).2.(1)Cell cycle analysis result showed that ESCC cell lines were arrested in G2/M phase(P< 0.05).(2)Western Blot result showed that P27 expression was increased in a dose dependent manner after treating with harmaline.3.(1)Kinase screen result showed that mTOR activity was decreased by 40% after treating with 20 m M harmaline,while other kineses did not show significantly change.(2)Western Blot result showed that p-mTOR(S2481),p-AKT(S473),p-p70S6k(T389)and p-GSK3β(S9)decreased by harmaline in a dose dependent manner.(3)In vitro kinase assay result showed that the activity of mTOR was decreased by harmaline in a dose dependent manner(P< 0.05).4.(1)Western Blot showed that the expression of mTOR was more heigher in ESCC cell lines compared with SHEE.After ESCC cell lines were infected with shmTOR,the expression of mTOR was decreased in ESCC knock down cell lines.(2)MTT,Foci-formation and Soft agar results showed that the cell growth was decreased in shmTOR cell lines.5.(1),(2)and(3)these phenotype assay result showed that MTT assay showed that shmTOR cell lines exhibit less sensitive to harmaline compared with parent cells.6.(1)Harmaline can inhibit tumor growth(P< 0.05),and don’t affect the bodyweight of treatment mice.(2)KI-67 expression was decreased after treated with hamaline(P< 0.05).(3)Target molecular expression was decreased by harmaline.(4)HE staining showed that there was not significant different for liver,spleen,kidney in group and control group.(5)ALT and AST level in the blood were similar in harmaline group and control group.It meaned that liver founction was not affected by harmaline.Conclusions: 1.Harmaline is a novel mTOR inhibitor and exhibit anti-tumor effect in vitro and in vivo.2.Harmaline strongly suppresses ESCC growth both in vivo and in vitro.3.Harmaline can inhibit ESCC tumor growth in vivo.